WO2021064774A1 - Polymère redox de poids moléculaire élevé et biocapteur l'utilisant - Google Patents

Polymère redox de poids moléculaire élevé et biocapteur l'utilisant Download PDF

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WO2021064774A1
WO2021064774A1 PCT/JP2019/038464 JP2019038464W WO2021064774A1 WO 2021064774 A1 WO2021064774 A1 WO 2021064774A1 JP 2019038464 W JP2019038464 W JP 2019038464W WO 2021064774 A1 WO2021064774 A1 WO 2021064774A1
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molecular weight
high molecular
polymer
linker
indicates
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PCT/JP2019/038464
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Japanese (ja)
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圭吾 羽田
和明 枝川
北脇　文久
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Ｐｈｃホールディングス株式会社
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Priority to EP19947973.4A priority Critical patent/EP4043499A4/fr
Priority to JP2021550738A priority patent/JP7285332B2/ja
Priority to PCT/JP2019/038464 priority patent/WO2021064774A1/fr
Priority to US17/764,842 priority patent/US20220322978A1/en
Priority to CN201980100881.5A priority patent/CN114450313A/zh
Publication of WO2021064774A1 publication Critical patent/WO2021064774A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/1486—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using enzyme electrodes, e.g. with immobilised oxidase
    • A61B5/14865—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using enzyme electrodes, e.g. with immobilised oxidase invasive, e.g. introduced into the body by a catheter or needle or using implanted sensors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
    • C12Q1/006—Enzyme electrodes involving specific analytes or enzymes for glucose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/14532—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue for measuring glucose, e.g. by tissue impedance measurement
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/14546—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue for measuring analytes not otherwise provided for, e.g. ions, cytochromes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/1486—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using enzyme electrodes, e.g. with immobilised oxidase
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
    • C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
    • C08G69/10—Alpha-amino-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/48—Polymers modified by chemical after-treatment
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
    • C08G73/02—Polyamines
    • C08G73/0206—Polyalkylene(poly)amines
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
    • Y02E60/30—Hydrogen technology
    • Y02E60/50—Fuel cells

Definitions

  • the present disclosure relates to a high molecular weight redox polymer and a biosensor using the same. More specifically, the present invention relates to an implantable biosensor in vivo.
  • a typical example of an electrochemical biosensor using an enzyme is an electrochemical glucose sensor used for self-blood glucose measurement.
  • Glucose oxidoreductase is used in the electrochemical glucose sensor.
  • These glucose oxidoreductases include glucose oxidase (Gox) and glucose dehydrogenase (GDH).
  • an implantable electrochemical glucose sensor that continuously or semi-continuously measures the glucose concentration in a living body has been developed.
  • the main body 10 is attached to the living body 2 and the probe 11 is inserted into the living body to continuously or semi-continuously in or between the blood. Measure the glucose concentration in the interstitial fluid.
  • the implantable electrochemical biosensor represented by such an implantable electrochemical glucose sensor inserts the probe into the living body for a long time (generally several days to several weeks). Therefore, the reagent layer containing the redox enzyme and the redox mediator (electron carrier) provided on the electrode is coated with a protective film.
  • a protective film Such a structure prevents or suppresses the opportunity for redox enzymes and redox mediators to flow out of the sensor. This is because if the redox enzyme or the redox mediator leaks out of the sensor, not only the measurement sensitivity is deteriorated, but also the durability of the sensor may be deteriorated, which may be unfavorable for the living body.
  • Patent Document 1 discloses an ionic hydrophilic high molecular weight redox polymer for an enzyme electrochemical sensor.
  • Patent Documents 2 to 6 disclose, for example, a phenazine-based compound having a substituent at the 5- or 1-position of phenazine (1-methoxy-5-methylphenazineium salt, etc.) as an electronic mediator of a biosensor.
  • Patent Document 7 discloses a phenazine-based compound having a substituent at the 5-position of phenazine or the like.
  • the implantable electrochemical biosensor it is important for the implantable electrochemical biosensor to take measures to prevent or suppress the outflow of redox enzymes and redox mediators from the reagent layer through the protective layer.
  • the outflow of redox mediator constituting the reagent layer can be further prevented or suppressed, and storage stability (durability) can be maintained while maintaining the measurement sensitivity of analite (glucose, etc.).
  • the challenge is to provide means that can be improved.
  • the present inventors have excellent performance of the phenazine-based compound as a redox mediator when a linker is introduced (derivatized) into the nitrogen atom at the 5-position of the phenazine-based compound.
  • Various high molecular weight polymers synthetic resin, protein, etc.
  • the linker can be linked via the linker while retaining the above, and the high molecular weight redox polymer thus obtained is contained in the reagent layer to be phenazine-based. It has been found that the outflow can be prevented or suppressed as compared with the case where the compound is contained alone in the reagent layer.
  • the present invention includes at least the following matters.
  • General formula (A1) wherein, X - represents an anion species, L is shown a linker, Poly represents a high molecular weight polymer, R 1 ⁇ R 8 are each independently a hydrogen atom, a halogen atom, a hydroxyl group, a carboxyl group , Amino group which may have a substituent, Saturated or unsaturated hydrocarbon group of a linear or branched chain having 1 to 6 carbon atoms which may have a substituent, and a substituent. Indicates a suitable acyl group, an alkoxy group which may have a substituent, or a phenyl group which may have a substituent.
  • the high molecular weight redox polymer has a general formula (B1): Wherein, X - represents an anion species, L is shown a linker, Poly represents a high molecular weight polymer, R 1 is a hydrogen atom, a halogen atom, a hydroxyl group, a carboxyl group, substituted A suitable amino group, a linear or branched saturated or unsaturated hydrocarbon group having 1 to 6 carbon atoms which may have a substituent, an acyl group which may have a substituent, and a substituent may be used.
  • the high molecular weight redox polymer represented by. [3] The high molecular weight redox polymer has a general formula (1): Wherein, X - represents an anion species, L is shown a linker, Poly represents a high molecular weight polymer. ] Item 3. The high molecular weight redox polymer according to Item 1 or 2. [4] The linker is any one of Items 1 to 3, wherein at least one atom selected from the group consisting of a carbon atom, a nitrogen atom, an oxygen atom and a sulfur atom is bonded in a chain shape to form a main chain.
  • the high molecular weight redox polymer described in. [5] The high molecular weight redox polymer has a general formula (2): [In the formula, X - indicates the anionic species, L 1 indicates the first linker, L 2 indicates the second linker, and Poly indicates the high molecular weight polymer. ] Item 4. The high molecular weight redox polymer according to any one of Items 1 to 4, which is represented by. [6] Item 5. The high molecular weight redox polymer according to Item 5, wherein the bond between the first linker and the second linker is a covalent bond. [7] Item 5.
  • the high molecular weight redox polymer has a general formula (4): [In the formula, X - indicates the anionic species, L 2 indicates the second linker, and Poly indicates the high molecular weight polymer. ] Or, general formula (5): [In the formula, X - indicates the anionic species, L 2 indicates the second linker, and Poly indicates the high molecular weight polymer. ] Or, general formula (6): [In the formula, X - indicates the anionic species, L 2 indicates the second linker, and Poly indicates the high molecular weight polymer.
  • Item 1 to 9 wherein in the high-molecular-weight polymer, at least one atom selected from the group consisting of a carbon atom, a nitrogen atom, an oxygen atom and a sulfur atom is bonded in a chain shape to form a main chain.
  • the high molecular weight redox polymer according to any one of the above.
  • Item 2 The high molecular weight according to Item 10, wherein the polymer polymer contains at least one selected from the group consisting of carbon-carbon bonds, amide bonds and ether bonds in the bonds between the atoms constituting the main chain. Redox polymer.
  • Item 2 The high molecular weight redox polymer according to Item 10 or 11, wherein the high molecular weight polymer is a protein, polypeptide or polynucleotide.
  • the anion species are halogen ion, ion of compound containing halogen, hydroxide ion, carboxylate ion, nitrate ion, nitrite ion, acetate ion, hydrogen carbonate ion, dihydrogen phosphate ion, hydrogen sulfate ion, alkyl.
  • Item 2 The high molecular weight redox according to any one of Items 1 to 131, which is any one selected from the group consisting of a sulfonate ion, a hydrogen sulfide ion, a hydrogen oxalate ion, a cyanate ion, and a thiocyanate ion. polymer.
  • Item 2 The high molecular weight redox polymer according to any one of Items 1 to 14, wherein the high molecular weight redox polymer is hydrophilic.
  • a high molecular weight redox polymer mixture comprising a plurality of types of the high molecular weight redox polymer according to any one of Items 1 to 15.
  • the reagent layer comprises a redox enzyme that oxidizes or dehydrogenates the analite, and the high molecular weight redox polymer according to any one of Items 1 to 15 or a mixture of the high molecular weight redox polymer according to Item 16.
  • Sensor. [18] Item 17. The biosensor according to Item 17, which has a reference electrode. [19] Item 6. The biosensor according to Item 17 or 18, wherein the oxidoreductase is a coenzyme-binding enzyme. [20] Item 6. The biosensor according to any one of Items 17 to 19, wherein the allite is glucose and the oxidoreductase is glucose oxidase or glucose dehydrogenase. [21] Item 20.
  • the biosensor according to Item 20 wherein the glucose dehydrogenase is a flavin adenine dinucleotide (FAD) -linked glucose dehydrogenase.
  • Item 2 The biosensor according to Item 20 or 21, wherein the glucose dehydrogenase has an enzyme activity against maltose of 5% or less when the enzyme activity against glucose is 100%.
  • Item 2 The biosensor according to Item 20 or 21, wherein the glucose dehydrogenase has an enzyme activity against maltose of 3% or less when the enzyme activity against glucose is 100%.
  • Item 2 The biosensor according to any one of Items 17 to 24, wherein the protective film has a hole through which an analyzer existing outside the protective film can permeate into the protective film.
  • the redox mediator phenazine derivative
  • the redox mediator flows out of the protective film (the biosensor is embedded in the living body). It is possible to further prevent or suppress the outflow into the living body at that time. As a result, storage stability (durability) can be improved while maintaining measurement sensitivity.
  • This is an example of a cross-sectional view of an implantable biosensor attached to a living body (human body). It is a top view of the front side of the implantable biosensor probe which is an example of the present disclosure.
  • It is sectional drawing in the AA'cutting line in FIG. It is sectional drawing in the B-B'cutting line in FIG.
  • the high molecular weight redox polymer of the present disclosure is a compound in which a phenazine-based compound, which is a redox mediator, is linked to the high molecular weight polymer via a linker bonded to the nitrogen atom at the 5-position thereof.
  • the "redox mediator” means a redox substance that mediates electron transfer, and in a biosensor, it refers to a substance that is responsible for the transfer of electrons generated by the redox reaction of an oxidoreductase by an oxidoreductase.
  • a "phenazine-based compound” having such a function, particularly a “phenazine derivative” in which a linker is introduced into the nitrogen atom at the 5-position thereof is used as the redox mediator.
  • Redox mediators include various compounds such as ferricyanides and ferrocene, but phenazine compounds have a redox potential lower than 0V (vs.Ag / AgCl saturated KCl), and ascorbic acid in biological samples. It is a particularly preferable redox mediator because it is not easily affected by electrochemical measurement impurities such as (vitamin C) and uric acid.
  • a "phenazine-based compound” is a compound having a phenazine skeleton (see the formula below) in which carbon atoms at positions 1 to 4 and 6 to 9 may be substituted, as long as it functions as a redox mediator. It is not particularly limited. Various types of such phenazine compounds are known, and can be used in the present invention for producing a high molecular weight redox polymer.
  • the high molecular weight redox polymer of the present disclosure can be represented by the general formula (A1).
  • X - represents an anion species
  • L is shown a linker
  • Poly represents a high molecular weight polymer.
  • R 1 to R 8 are independently substituted with, for example, (i) hydrogen atom, (ii) halogen atom, (iii) hydroxyl group, (iv) carboxyl group, and (v) substitution.
  • Amino group which may have a group, (vi) C 1-6 (1 to 6 carbon atoms, lower alkyl) which may have a substituent, which is a saturated or unsaturated linear or branched chain.
  • An alkoxy group) and (ix) indicate a phenyl group which may have a substituent.
  • substituents that each of the above (v), (vi), (vii), (viii) and (ix) may have include (a) a halogen atom, (b) a hydroxyl group and (c).
  • Carboxyl group (d) amino group, (e) C 1-6 (1-6 carbon atoms, lower alkyl) linear or branched saturated or unsaturated hydrocarbon group, (f) acyl group (eg) C 1-6 alkyl-carbonyl group), (g) alkoxy group (eg C 1-6 alkoxy group), (h) carboxyl-alkyl group (eg carboxyl-C 1-6 alkyl group), (i) mesyl group, (K) Examples include phenyl groups.
  • the number of substituents that each of the above (v), (vi), (vii), (viii) and (ix) may have is not particularly limited, and is, for example, one, two or three.
  • R 1 to R 8 are not limited to the above-mentioned substituents, and may be substituents having a known phenazine-based compound at each site.
  • the general formula (A1) is apparently expressed as having only one phenazine derivative bonded to one high molecular weight polymer (Poly), but it is interpreted in such a limited manner. It should be interpreted that at least one, usually multiple phenazine derivatives, are bound to one high molecular weight polymer. Further, in the production method for producing a high molecular weight redox polymer by reacting a phenazine derivative with a high molecular weight polymer as described in the present specification, the number of bonds of the phenazine derivative to one high molecular weight polymer has a distribution and is high. A molecular weight polymer composition is obtained.
  • the distribution method of the number of phenazine derivatives bonded to the high molecular weight polymer composition and the number (average) of the phenazine derivatives bonded to one high molecular weight polymer are not particularly limited, and the application and performance are not particularly limited. It can be appropriately adjusted depending on the raw material (high molecular weight polymer or the monomer constituting the high molecular weight polymer) and the reaction conditions when producing the high molecular weight redox polymer. For example, the number of the first or second reactive groups shown in Table 1 below may be increased or decreased in one molecule of the monomer used as a raw material of the high molecular weight polymer (such as the portion of the 2'linker), or the monomer may be contained in the whole monomer.
  • the ratio of those reactive groups that actually react with the corresponding reactive groups contained in the phenazine derivative may be increased or decreased (average) of the number of bonds of the phenazine derivative.
  • the distribution method can be changed, and a high molecular weight redox polymer (composition) having desired properties, in which a desired number of phenazine derivatives are bonded to the high molecular weight polymer, can be produced. ..
  • the anion species is not particularly limited, but for example, halogen ion, ion of a compound containing halogen, hydroxide ion, carboxylate ion, nitrate ion, nitrite ion, acetate ion, hydrogen carbonate ion, phosphorus. Any one selected from the group consisting of dihydrogen acid ion, hydrogen sulfate ion, alkyl sulfonic acid ion, hydrogen sulfide ion, hydrogen oxalate ion, cyanate ion, and thiocyanate ion can be mentioned.
  • the high molecular weight redox polymer of the present disclosure is hydrophilic.
  • the hydrophilic high-molecular-weight redox polymer referred to here is a polymer that has a high affinity for water and is easily dissolved and miscible in water and a polar solvent, preferably a polymer used as a reaction solvent with a phenazine derivative. It refers to a polymer that is easily dissolved and miscible to the extent that it does not interfere with the progress of the reaction.
  • Types of polar solvents include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-1-propanol, 2-methyl-2-propanol, formic acid, acetic acid, tetrahydrofuran, and acetone. , Dioxane, methyl tylketone, ethyl acetate, acetonitrile, dimethylformamide, dimethyl sulfoxide and the like.
  • the high molecular weight redox polymer is a phenazine derivative and a high molecular weight polymer bonded via a linker, and is a phenazine derivative (including a first'linker portion described later) and a high molecular weight polymer (a second'linker portion described later). Either one of them may be hydrophobic. That is, the finally synthesized high molecular weight redox polymer may be hydrophilic.
  • At least one atom selected from the group consisting of a carbon atom, a nitrogen atom, an oxygen atom and a sulfur atom is bonded in a chain form to form a main chain.
  • a ether bond, a thioether bond, an amide bond, or the like is formed in the middle as a group generated by the reaction of the first reaction group and the second reaction group described later, or a group formed in advance instead. May be included as a hydrocarbon group.
  • the main chain formed by "at least one atom selected from the group consisting of carbon atom, nitrogen atom, oxygen atom and sulfur atom is bonded in a chain shape" is a linear ring (cyclic type). Not only when it is composed of a chain compound (acyclic compound) which refers to a compound having a molecular structure without any structure derived from the compound (cyclic structure), but also when it is composed of a compound which partially has a ring. It is also included if there is.
  • one of the rings for example, on one carbon atom of the benzene ring
  • the other for example, the linear chain
  • one chain compound side binds to the phenazine derivative (for example, the alkyl group becomes an alkylene group), and the other chain compound side binds to the high molecular weight polymer.
  • One or both of the above two groups derived from an open chain compound attached across a ring can also be replaced with a group derived from a partially ringed compound.
  • the linker may have a side chain bonded to the main chain.
  • linker is not only as an explanation of “L” in the general formula (A1), but also as “L 1 " (first linker) and “L 2 " in the general formula (A2) described below. It is also applied as an explanation of each of the second linkers) and “L 1 -L 2 " to which they are bound, and further as an explanation of "L 2" (second linker) in the general formulas (A3) to (A9).
  • L 1 and L 2 are different types of linkers, for example, linkers having different repeating units.
  • the number of atoms (total of carbon atom, nitrogen atom, oxygen atom and sulfur atom) constituting the main chain constituting the main chain constituting Examples (A3) to (A9) is preferably 10 or more, and 31 The above is preferable, and 55 or more is more preferable.
  • the number of atoms constituting the main chain constituting the linker is preferably less than 720, preferably less than 340, and even more preferably less than 240.
  • the high molecular weight redox polymer represented by the general formula (A1) is preferably represented by the general formula (B1).
  • Each symbol in the general formula (B1) is synonymous with the corresponding symbol in the general formula (A1).
  • R 7 to R 8 are all hydrogen atoms (unsubstituted), and only R 1 is a hydrogen atom (unsubstituted).
  • a predetermined substituent may be used.
  • the high molecular weight redox polymer represented by the general formula (A1) or (B1) is particularly preferably represented by the general formula (1).
  • each symbol in the general formula (1) is synonymous with the corresponding symbol in the general formula (A).
  • the general formula (1) defines in the general formula (A1) that R 7 to R 8 are all hydrogen atoms (unsubstituted) and R 1 is a methoxy group (alkoxy group). Corresponds to the embodiment.
  • the linker contained in the high molecular weight redox polymer of the present disclosure is preferably one in which two types of linkers, a first linker and a second linker are bonded.
  • Ultra-high molecular weight redox polymers having such a linker replace the linker (L) with one in which the first linker and the second linker are bonded (-L 1 -L 2-), for example, in the general formula (A1). It can be expressed as a general formula (A2).
  • L 1 indicates the first linker
  • L 2 indicates the second linker
  • each other symbol is synonymous with each corresponding symbol in the general formula (A1).
  • the site (L 1 -L 2 ) to which the first linker and the second linker are bound in the general formula (A2) is a site corresponding to the linker (L) in the general formula (A1).
  • first linker and the second linker are preferably bound by a covalent bond.
  • first linker and the second linker may be bound by a non-covalent bond (for example, electrostatic interaction) as long as the effects of the present invention are exhibited.
  • the covalent bond formed at the binding site between the first linker and the second linker is not particularly limited, but for example, the first reactive group (such as the amine shown in the table) shown in Table 1 below is used.
  • a monovalent group derived from a compound, particularly an amino group at the terminal thereof, and a second reactive group a monovalent group derived from a compound such as a carboxylic acid shown in the table, particularly a carboxyl at the terminal thereof).
  • Examples include covalent bonds formed by the reaction of (groups, etc.). Such covalent bonds and reactive groups for forming them are well known and customary to those skilled in the art (eg, JP 2003-514924).
  • the first and second reactive groups shown in Table 1 are a first linker before binding (hereinafter sometimes referred to as “first'linker”) and a second linker before binding (hereinafter “second'linker”). ”), One of the first'linker and the second'linker has a first reactive group, and the other has a second reactive group.
  • the second linker (L 2 ) in the general formula (2) and the general formulas (3) to (9) described later is an atom of a binding site generated by the reaction of the first reaction group and the second reaction group, and the reaction thereof. Contains atoms in sites that have not changed due to.
  • an amine (amino group: -NH 2 ) is present as a first reactive group at the end of the 1'linker of the phenazine derivative, and as a second reactive group at the end of the 2'linker of the high molecular weight polymer.
  • a carboxylic acid (carboxyl group: -COOH) is present and they react to form an amide bond (-NH-CO-)
  • the second linker represented by L 2 in the general formula (3) is , -NH-CO- contains atoms.
  • the phenazine derivative and the high molecular weight polymer are linked by an amide bond, that is, the first'linker or other site of the phenazine derivative and the second of the high molecular weight polymer.
  • the linker or other site is linked by an amide bond formed by the reaction of the first reactive group (amine) corresponding to the "amide” in Table 1 with the second reactive group (carboxylic acid, activated ester, etc.). Has been done.
  • the phenazine derivative and the high molecular weight polymer are linked by a covalent bond other than an amide bond, that is, the first'linker or other site of the phenazine derivative and the high molecular weight polymer.
  • the second'linker or other moiety contained in is "ether”, “thioether”, “ester”, “thioester”, “hydrazone”, “urea”, “thiourea”, “oxime”, in Table 1.
  • the first and second reactive groups shown in Table 1 are those contained in the side chains of the amino acid residues constituting the protein or the like. It may be the one possessed by the linker (second'linker) separately introduced into the protein.
  • linker second'linker
  • lysine, arginine and histidine have an "amine” (amino group) as the "first reactive group”
  • aspartic acid and glutamic acid have "first reactive group” or "second reactive group” as "second reactive group”.
  • cysteine corresponds to an amino acid having a "thiol” (sulfanyl group) as the "first reactive group”. Therefore, a protein or polypeptide containing those amino acid residues has a first or second reactive group, respectively, and reacts with a predetermined corresponding reactive group of a phenazine derivative under predetermined reaction conditions. Can form a covalent bond.
  • a polynucleotide when used as the high molecular weight polymer, it may be possessed by an unnatural modified nucleotide used as a part of the nucleotides constituting the polynucleotide, or a polynucleotide may be separately provided by other means. It may be the one possessed by the linker (second'linker) introduced in.
  • linker second'linker
  • the first linker preferably constitutes the main chain as described above, and examples thereof include those containing at least one of a polyethylene glycol chain and a hydrocarbon chain.
  • the first linker in the general formula (2) can be a linker as represented by a site corresponding to the first linker in the following general formulas (3) to (9).
  • high molecular weight redox polymer represented by the general formula (A2) include those represented by the general formula (A3).
  • high molecular weight redox polymer represented by the general formula (A3) include those represented by any of the general formulas (A4) to (A9).
  • L 2 indicates the second linker, and the other symbols are synonymous with the corresponding symbols in the general formula (A1).
  • L 2 indicates the second linker, and the other symbols are synonymous with the corresponding symbols in the general formula (A1).
  • L 2 indicates the second linker, and the other symbols are synonymous with the corresponding symbols in the general formula (A1).
  • L 2 indicates the second linker, and the other symbols are synonymous with the corresponding symbols in the general formula (A1).
  • L 2 indicates the second linker, and the other symbols are synonymous with the corresponding symbols in the general formula (A1).
  • L 2 indicates the second linker
  • n indicates an integer from 1 to 80, for example, 1 to 30, and each other symbol is a corresponding symbol in the general formula (A1). Is synonymous with.
  • the respective linkers (L) are replaced with those in which the first linker and the second linker are bonded (-L 1 -L 2- ).
  • (B2) and (2) can be defined as high molecular weight redox polymers corresponding to the general formula (A2).
  • the polymer redox polymers corresponding to the general formula (A3) given as a specific example of the general formula (A2) and the general formulas (A4) to (A9) given as a specific example of the general formula (A3) are also described above.
  • the general formulas (B3) to (B9) and the general formulas (3) to (9) can be specified.
  • the high molecular weight polymer in the present disclosure is bound to the redox mediator in order to prevent or suppress the outflow of the redox mediator (phenazine derivative) from the protective film. Therefore, as the high molecular weight polymer, a polymer having a molecular weight (weight average molecular weight) of a certain level or more, usually a polymer having a weight average molecular weight of 10,000 or more, preferably 50,000 or more, more preferably 100,000 or more is used.
  • the weight average molecular weight of this high molecular weight polymer is usually less than 10,000,000, preferably less than 1,000,000.
  • the molecular weight (weight average molecular weight) and molecular weight distribution of the high molecular weight polymer can be measured by known means according to the type of the high molecular weight polymer. For example, gel permeation chromatography (GPC) or high molecular weight polymer can be used. If it is a protein, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) or the like can be used. When a commercially available high molecular weight polymer is purchased and used, the numerical value shown in the catalog value or the like can be regarded as the molecular weight (weight average molecular weight) or the like.
  • GPC gel permeation chromatography
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • the high molecular weight polymer may be a homopolymer, a copolymer, and a polymer in which they are bonded and / or mixed, and may be a random polymer or a block polymer. May be good.
  • the high molecular weight polymer has a structure capable of being bonded to a phenazine derivative, that is, a redox mediator (phenazine-based compound, particularly the nitrogen atom at the 5-position thereof) via a linker, and a reagent layer containing the redox mediator on the working electrode.
  • the polymer is not particularly limited as long as it is a high-molecular-weight polymer capable of forming the above.
  • a high-molecular-weight polymer for example, a polymer in which at least one atom selected from the group consisting of carbon atom, nitrogen atom, oxygen atom and sulfur atom is bonded in a chain shape to form a main chain is typical.
  • Examples include those containing at least one selected from the group consisting of carbon-carbon bonds, amide bonds and ether bonds in the bonds between the atoms constituting the main chain.
  • the high molecular weight polymer may have a side chain bonded to the main chain as described above. It suffices if it can be linked to at least one phenazine derivative in the main chain (longitudinal end) and / or side chain of the high molecular weight polymer.
  • the carbon-carbon bond is a bond derived from an ethylenic carbon-carbon double bond, which is typically contained in the ethylene-based polymer described later.
  • the amide bond is typically a bond (peptide bond) contained in the polyamino acid-based polymer described later.
  • the high molecular weight polymer is linked with multiple (many) phenazine derivatives in the side chain.
  • a second'linker having a first or second reactive group as shown in Table 1 above, which can react with the first'linker of the phenazine derivative to form a covalent bond is sided.
  • a high molecular weight polymer having a plurality (many) as a chain is preferable.
  • the first or second reactive group may be located at the end of a second'linker composed of a relatively large number of atoms introduced, for example by an addition reaction to the main chain, or the main chain. Due to the original possession of the monomer used to form it, it may be directly attached to the main chain or located at the end of a 2'linker composed of a relatively small number of atoms. Good.
  • high molecular weight polymer in the present disclosure include polyamino acid-based polymers (for example, poly (L-sodium L-glutamate), poly (L-lysine)); polyimine-based polymers (for example, poly (ethyleneimine)); or ethylene-based polymers.
  • polyamino acid-based polymers for example, poly (L-sodium L-glutamate), poly (L-lysine)
  • polyimine-based polymers for example, poly (ethyleneimine)
  • ethylene-based polymers for example, polyallylamine hydrochloride, allylamine hydrochloride / diallylamine hydrochloride copolymer, allylamine / diallyldimethylammonium chloride copolymer
  • These monomers include homopolymers and copolymers and their modifications (for example, vinyl acetate-derived units converted to vinyl alcohol by saponification, or treated to impart hydrophilicity). ..
  • Examples of the monomer containing an ethylenic carbon-carbon double bond include ethylene, propylene, butadiene, isobutene, tetrafluoroethylene, vinyl alcohol, vinyl acetate, vinyl chloride, vinylidene chloride, styrene, methylstyrene, allylamine, diallylamine, and diallyl. Examples thereof include dimethylammonium chloride, acrylic acid, methacrylic acid, methyl acrylate, methyl methacrylate, acrylonitrile and the like.
  • various high molecular weight polymers generally known as biocompatible polymers, for example, polyester-based polymers such as polyethylene terephthalate can be used.
  • a high-molecular-weight polymer can be said to be a polymer using an amino acid as a monomer (however, it is conceptually distinguished from the above-mentioned polyamino acid), and has a natural amino acid sequence or a modified (substituted, deleted, added, etc.) amino acid. It may be a protein or polypeptide having a sequence. Examples of such proteins include BSA and the like, which are well-known and commonly used as blocking agents, and oxidoreductases in the present disclosure such as glucose dehydrogenase and glucose oxidase.
  • the high molecular weight polymer is a polynucleotide having a natural base sequence or a modified (substituted, deleted, added, etc.) base sequence, which can be said to be a polymer in which nucleotides are used as monomers and linked via a phosphate group. There may be.
  • the high molecular weight polymer has hydrophilicity.
  • the high molecular weight redox polymer needs to be hydrophilic, while the high molecular weight polymer does not necessarily have to be hydrophilic. This is because even a non-hydrophilic high molecular weight polymer may be combined with a hydrophilic phenazine derivative to produce a high molecular weight redox polymer that is hydrophilic as a whole.
  • the high molecular weight polymer and the phenazine derivative are typically bonded in a reaction solvent containing water or a polar solvent in which the phenazine derivative is dissolved (for example, the first reaction group and the second reaction group are reacted). , It is desirable that the high molecular weight polymer is hydrophilic.
  • the high molecular weight redox polymer according to the present disclosure can be produced based on the technical level of those skilled in the art.
  • a high molecular weight redox polymer can be obtained by reacting a pre-synthesized phenazine derivative with the high molecular weight polymer according to the target high molecular weight redox polymer.
  • the phenazine derivative comprises a selected phenazine-based compound and a compound capable of forming a linker having a desired reactive group (for example, an N-alkylating agent) under appropriate conditions (temperature, time, auxiliary agent, etc.). It is obtained by introducing a linker (1st'linker) into the nitrogen atom at the 5-position of the phenazine-based compound by reacting with.
  • a linker (1st'linker
  • a high molecular weight polymer having a desired reactive group can be obtained by polymerizing the selected monomer by an appropriate method (reaction, condition, auxiliary agent, etc.) or by synthesizing it as a protein or nucleic acid. Be done.
  • the target high molecular weight redox polymer is obtained by reacting the phenazine derivative and the high molecular weight polymer under appropriate conditions according to the reactive groups (the first and second reactive groups in Table 1) of each. Is obtained.
  • the obtained high molecular weight redox polymer may be purified by gel filtration chromatography, ultrafiltration or the like, if necessary.
  • any one of them may be used alone. Alternatively, two or more kinds may be used in combination, for example, mixed.
  • a plurality of types of high molecular weight redox polymers of the present disclosure eg, different types of high molecular weight polymers, different types of linkers, types of phenazine compounds (position, number of substituents, etc.
  • High molecular weight redox polymer mixtures are provided that include (such as different types).
  • the biosensor for detecting or quantifying an analysis of the present disclosure is basically an implantable type and covers a working electrode, a counter electrode, a reagent layer arranged on the working electrode, and at least the reagent layer.
  • the reagent layer has a protective film, and the reagent layer comprises a redox enzyme that oxidizes or dehydrogenates the analysis, and a high-molecular-weight redox polymer according to the present disclosure or a mixture thereof, typically the general formula (1):
  • the description of each symbol in the general formula (A1) is as described above. ] Includes a high molecular weight redox polymer represented by or a mixture containing a plurality of types of the high molecular weight redox polymer represented by the general formula (A1).
  • the reagent layer of the biosensor may contain any one kind of high molecular weight redox polymer alone, or two or more kinds of high molecular weight redox polymers may be mixed or laminated (for example, high molecular weight redox). May include (as a polymer mixture).
  • the biosensor of the present disclosure may further include a reference electrode.
  • oxidoreductase refers to an enzyme capable of oxidizing or dehydrogenating an analysis.
  • This redox enzyme is preferably a coenzyme-binding enzyme.
  • the oxidoreductase is glucose oxidase (glucose oxidase) or glucose dehydrogenase (glucose dehydrogenase; GDH).
  • glucose oxidase glucose oxidase
  • GDH glucose dehydrogenase
  • coenzyme-bound glucose dehydrogenase include pyrroloquinoline quinone (PQQ) -linked GDH and flavin adenine nucleotide (FAD) -linked GDH.
  • PQQ pyrroloquinoline quinone
  • FAD flavin adenine nucleotide
  • GDH has an enzyme activity on maltose of 5% or less when the enzyme activity on glucose is 100%. More preferably, the enzyme activity against maltose is 3% or less.
  • FAD-bound GDH An example of an enzyme having such enzyme activity is FAD-bound GDH.
  • FAD-bound GDH include those derived from the genus Aspergillus (Orize and Terreus) and the genus Mucor (for example, WO2004 / 058958, Japanese Patent Application Laid-Open No. 2008-228740, WO2010 / 140431). See publication).
  • the redox enzyme in the present disclosure can also be cross-linked with a high-molecular-weight redox polymer using a cross-linking agent such as glutaraldehyde.
  • a cross-linking agent such as glutaraldehyde.
  • the redox enzyme maintains measurable activity of the analysis even if it is cross-linked.
  • the redox enzyme may be contained in the reagent layer in a state of being mixed with the high molecular weight redox polymer.
  • the "protective film” is a substance that prevents or suppresses leakage of substances (mainly redox mediators) contained in the reagent layer to the outside of the protective film, and an analog that exists outside the protective film. It has permeable pores in the protective membrane in which the reagent layer is present. Then, the protective film needs to be arranged so as to cover at least the reagent layer on the probe.
  • substances mainly redox mediators
  • the protective film covering the surface thereof is biocompatible with which proteins and cells are not easily adsorbed.
  • the protein is formed of a polymer having such properties.
  • the polymer capable of forming such a protective film include a copolymer of methyl methacrylate and hydroxyethyl methacrylate, a copolymer of butyl methacrylate and hydroxyethyl methacrylate, and poly (2-methacryloyloxyethyl phosphorylcholine-conn-butyl methacrylate). ) Etc. can be mentioned.
  • a (meth) acrylate-based compound having a main chain similar to that of these exemplified polymers and capable of reacting with a linker (first or second reactive group shown in Table 1) is used.
  • Those having a side chain can also be used as an "ethylene-based polymer" having a methacryloyl group or an acryloyl group mentioned as a specific example of a high molecular weight polymer.
  • Figures 3 to 6 show an example of the internal structure of the probe of the implantable biosensor to which the high molecular weight redox polymer of the present disclosure is applied.
  • the configurations shown in FIGS. 3 to 6 are exemplary and do not limit the application of the high molecular weight redox polymer of the present disclosure.
  • the implantable biosensor 1 includes a main body 10 and a probe 11.
  • FIG. 3 shows a top view of the probe 11 as viewed from the front side.
  • the front side referred to here refers to the surface on the side having the working electrode and the reference electrode described later.
  • the probe 11 is roughly composed of a sensing portion 121 to be inserted into the living body and a terminal portion 122 for electrically connecting to the internal circuit of the biosensor main body 10.
  • the sensing portion 121 is formed thin so that it can be inserted into the living body.
  • the sensing portion is to be inserted into the body, and generally has a length of 20 to 3 mm (preferably 10 to 3 mm) in the longitudinal direction and a length of 1 mm to 50 ⁇ m (preferably 500 ⁇ m to 50 ⁇ m) in the lateral direction. It is preferably configured.
  • FIG. 4 shows a cross-sectional view of the probe 11 at the AA'cut line of FIG.
  • a metal thin film is formed on both sides of the insulating substrate 111 by a conductive metal material selected from the group consisting of carbon or a metal such as gold, silver, platinum or palladium by a sputtering method, a vapor deposition method, ion plating or the like.
  • the conductive thin film 112 is formed.
  • a groove 113 having a depth reaching the surface of the insulating substrate 111 is formed by drawing a laser on the conductive thin film 112 formed on one surface (front side) of the insulating substrate 111, and is referred to as a working electrode region 112a. It is electrically insulated by separating it into the electrode region 112b.
  • the insulating resist films 116a and 116b are formed on both sides of the insulating substrate 111 so as to have openings (openings for the reference electrode) at predetermined positions of the reference electrode region 112b.
  • the insulating resist films 116a and 116b are not formed up to a portion separated from the end portion of the sensing portion 112 by a certain distance.
  • the region where the resist film is not formed on the end side of the working electrode region 112a becomes the working electrode 114, and the resist film is not formed on the opposite side (the back side here).
  • the area is the counter electrode 117.
  • the reference electrode 115 is formed by forming Ag / AgCl in the opening for the reference electrode of the insulating resist film 116a formed on the front side of the insulating substrate 111 by a screen printing method or an inkjet method. Although an example of a three-electrode type of a working electrode, a counter electrode, and a reference electrode is shown here, a two-electrode type of a working electrode and a counter electrode may be used.
  • the reagent layer 118 contains at least a high-molecular-weight redox polymer in which a high-molecular-weight polymer and a redox mediator are bound, and an oxidoreductase, and is formed on the working electrode 114.
  • the reagent layer 118 is formed by, for example, applying and drying a solution containing a high molecular weight redox polymer and an oxidoreductase of an analysis (for example, glucose) on the working electrode 114.
  • the reagent layer 118 can also contain other components such as conductive particles such as carbon particles and a buffer solution component.
  • the reagent layer 118 can be formed by adding another component to a solution containing a high molecular weight redox polymer and an oxidoreductase, and applying and drying the component.
  • the high molecular weight redox polymer may be one in which the redox enzyme of Analite is bound to the redox mediator as a high molecular weight polymer (protein).
  • the high molecular weight redox polymer (the high molecular weight polymer contained therein is not the redox enzyme of Analite) may be crosslinked with the redox enzyme of Analite.
  • the reagent layer 118 may contain at least a high molecular weight redox polymer, and it is not necessary to separately contain an additional redox enzyme.
  • a protective film 119 can be formed on both sides, side surfaces and the tip portion of the sensing portion 121. It can be used as a probe 11.
  • FIG. 5 shows a cross-sectional view taken along the B-B'cutting line in FIG. 4, and FIG. 6 shows a cross-sectional view taken along the C-C' cutting line in FIG.
  • the protective film 119, the reagent layer 118, the working electrode 114, the insulating substrate 111, the counter electrode 117, and the protective film 119 are formed in this order from the front side to the back side (in the direction of the arrow in the drawing). Further, as shown in FIG.
  • the protective film 119 from the front side to the back side (in the direction of the arrow in the figure), the protective film 119, the reference electrode 115 (insulating resist film 116a), the working electrode region 112a, the insulating substrate 111, and the counter electrode The region 112c, the insulating resist film 116b and the protective film 119. Since the working electrode region 112a and the counter electrode region 112c shown in FIG. 6 are provided with the insulating resist films 116a and 116b on the upper surface thereof, they do not function as the working electrode and the counter electrode.
  • the general formula of the high molecular weight redox polymer and the high molecular weight polymer used for producing the high molecular weight redox polymer shown in the examples applies to all the repeating units (structures enclosed in parentheses) derived from each monomer constituting the high molecular weight polymer. It is expressed as if the phenazine derivative is bound. However, this expression is a measure for convenience, and in the actual high molecular weight redox polymer, the phenazine derivative is not necessarily bound to all the repeating units derived from each monomer constituting the high molecular weight polymer depending on the reaction conditions at the time of production. May be good. It is confirmed from the result of the "evaluation test" that the various high molecular weight redox polymers produced in the examples can carry out the invention according to the present disclosure.
  • Example 1 Production of PGA-C24-Ph
  • a phenazine derivative 6.47 mg of 5- ⁇ 12-[(12-ammoniododecyl) oxy] dodecyl ⁇ -1-methoxyphenazine-5-ium dinitrate (Ph-C24-NH3 +) (manufactured by Tokyo Chemical Industry Co., Ltd.) It was weighed and dissolved in 500 ⁇ L of ethanol.
  • poly (sodium L-glutamate) represented by the general formula (11a) (Peptide Institute, Ltd. Code 3063; MW> 12000, cutoff by dialysis) is 11.86 mg. It was weighed and dissolved in 1.5 mL of 100 mM 2-morpholinoetan sulfonic acid (MES) buffer (pH 6.0). Separately, 8.8 mg of water-soluble carbodiimide (WSC) (Dojin Kagaku) was weighed and dissolved in 500 ⁇ L of 100 mM MES buffer (pH 6.0). The above three solutions were mixed and reacted at room temperature for 4 hours with stirring.
  • MES 2-morpholinoetan sulfonic acid
  • WSC water-soluble carbodiimide
  • the reaction solution was subjected to gel filtration chromatography using a PD-10 column (GE Healthcare) using phosphate buffered saline (PBS, pH 7.4) as an elution buffer.
  • the solution after gel filtration was subjected to ultrafiltration using a centrifugal ultrafiltration filter (Amicon Ultra-4 10k; Merck Millipore).
  • PGA-C24-Ph high molecular weight redox polymer in which phenazine was covalently bonded to poly (sodium L-glutamate) represented by the general formula (5a) was obtained.
  • the obtained solution of PGA-C24-Ph is diluted 20 times, and the diluted solution is 386 nm using a microplate (greiner bio-one UV-STAR MICROPALLETE 96 WELL F-BODEN) and a plate reader (TECAN infinite M200 PRO). Dilution was measured. Then, the concentration of the PGA-C24-Ph solution was adjusted with PBS so that the absorbance at 386 nm was about 0.55 when the PGA-C24-Ph solution was diluted 20-fold.
  • the peak appearing at the absorbance at 386 nm is the peak derived from the phenazine derivative (the peak derived from the phenazine derivative has the maximum peak at about 384 to 386 nm). As the absorbance, the value obtained by subtracting the measured absorbance of PBS as a blank value was used.
  • Example 2 Production of PLL-C11-Ph
  • a phenazine derivative 5- ⁇ 11-[(2,5-dioxopyrrolidin-1-yl) oxy] -11-oxoundecyl ⁇ -1-methoxyphenazine-5-ium nitrate (Ph-C11-Su) (Tokyo Kasei Kogyo) 0.70 mg (manufactured by Co., Ltd.) was weighed and dissolved in 500 ⁇ L of 100 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 6.0).
  • MES 2-morpholinoethanesulfonic acid
  • poly (L-lysine) hydrochloride represented by the general formula (11b) (Peptide Institute Co., Ltd. Code 3075; MW> 12000, cutoff by dialysis) is 3.34. mg Weighed and dissolved in 500 ⁇ L of 100 mM MES buffer (pH 6.0). The above two solutions were mixed and reacted at room temperature for 4 hours with stirring.
  • the reaction solution was subjected to gel filtration chromatography using a PD-10 column (GE Healthcare) using PBS as an elution buffer.
  • the solution after gel filtration was subjected to ultrafiltration using a centrifugal ultrafiltration filter (Amicon Ultra-4 30k; Merck Millipore).
  • the obtained solution of PLL-C11-Ph is diluted 20 times, and the diluted solution is 386 nm using a microplate (greiner bio-one UV-STAR MICROPALLETE 96 WELL F-BODEN) and a plate reader (TECAN infinite M200 PRO). Dilution was measured. Then, when the solution of PLL-C11-Ph was diluted 20-fold, the concentration of the solution of PLL-C11-Ph was adjusted with PBS so that the absorbance at 386 nm was about 0.55. As the absorbance, the value obtained by subtracting the measured absorbance of PBS as a blank value was used.
  • the reaction solution was subjected to gel filtration chromatography using a PD-10 column (GE Healthcare) using PBS as an elution buffer.
  • the solution after gel filtration was subjected to ultrafiltration using a centrifugal ultrafiltration filter (Amicon Ultra-4 30k; Merck Millipore).
  • the obtained solution of PLL-C5-Ph_1 is diluted 20 times, and the diluted solution is 386 nm using a microplate (greiner bio-one UV-STAR MICROPALLETE 96 WELL F-BODEN) and a plate reader (TECAN infinite M200 PRO). Dilution was measured. Then, the concentration of the solution of PLL-C5-Ph_1 was adjusted with PBS so that the absorbance at 386 nm was about 0.55 when the solution of PLL-C5-Ph_1 was diluted 20-fold. As the absorbance, the value obtained by subtracting the measured absorbance of PBS as a blank value was used.
  • the reaction solution was subjected to gel filtration chromatography using a PD-10 column (GE Healthcare) using 10 mM of sodium phosphate buffer (pH 6.5) as an elution buffer.
  • the solution after gel filtration was subjected to ultrafiltration using a centrifugal ultrafiltration filter (Amicon Ultra-4 10k; Merck Millipore).
  • the obtained PAA-C5-Ph solution is diluted 20-fold, and the diluted solution is 386 nm using a microplate (greiner bio-one UV-STAR MICROPALLETE 96 WELL F-BODEN) and a plate reader (TECAN infinite M200 PRO). Dilution was measured. Then, the concentration of the PAA-C5-Ph solution was adjusted with PBS so that the absorbance at 386 nm was about 0.55 when the PAA-C5-Ph solution was diluted 20-fold. For the absorbance, a value obtained by subtracting the measured absorbance of 10 mM of sodium phosphate buffer (pH 6.5) as a blank value was used.
  • Example 5 Production of PEI-C5-Ph
  • a phenazine derivative 5- ⁇ [(2,5-dioxopyridine-1-yl) oxy] -5-oxopentyl ⁇ -1-methoxyphenazinenium nitrate (Ph-C5-Su) (Tokyo Kasei Kogyo Co., Ltd.) 2.38 mg was weighed and dissolved in 1 mL of 100 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 6.0).
  • MES 2-morpholinoethanesulfonic acid
  • a poly (ethyleneimine) solution represented by the general formula (11d) (SIGMA-ALDRICH product number 181978; number average molecular weight Mn ⁇ 60,000 measured by GPC, weight average measured by LS).
  • Molecular weight Mw ⁇ 750,000, 50% by weight in H2O was measured in 5 mg and dissolved in 1.5 mL of 100 mM MES buffer (pH 6.0). The above two solutions were mixed and reacted at room temperature for 4 hours with stirring.
  • the reaction solution was subjected to gel filtration chromatography using a PD-10 column (GE Healthcare) using PBS as an elution buffer.
  • the solution after gel filtration was subjected to ultrafiltration using a centrifugal ultrafiltration filter (Amicon Ultra-4 30k; Merck Millipore).
  • the obtained solution of PEI-C5-Ph is diluted 20 times, and the diluted solution is 386 nm using a microplate (greiner bio-one UV-STAR MICROPALLETE 96 WELL F-BODEN) and a plate reader (TECAN infinite M200 PRO). Dilution was measured. Then, the concentration of the PEI-C5-Ph solution was adjusted with PBS so that the absorbance at 386 nm was about 0.55 when the PEI-C5-Ph solution was diluted 20-fold. As the absorbance, the value obtained by subtracting the measured absorbance of PBS as a blank value was used.
  • the reaction solution was subjected to gel filtration chromatography using a PD-10 column (GE Healthcare) using PBS as an elution buffer.
  • the solution after gel filtration was subjected to ultrafiltration using a centrifugal ultrafiltration filter (Amicon Ultra-4 30k; Merck Millipore).
  • the obtained solution of PAA-DAA-C5-Ph is diluted 20 times, and the diluted solution is used with a microplate (greiner bio-one UV-STAR MICROPALLETE 96 WELL F-BODEN) and a plate reader (TECAN infinite M200 PRO).
  • the absorbance at 386 nm was measured.
  • the concentration of the PAA-DAA-C5-Ph solution was adjusted with PBS so that the absorbance at 386 nm was about 0.55 when the PAA-DAA-C5-Ph solution was diluted 20-fold.
  • the absorbance the value obtained by subtracting the measured absorbance of PBS as a blank value was used.
  • Example 7 Production of PAA-DADMA-C5-Ph
  • a phenazine derivative 2.04 mg of Ph-C5-Su (manufactured by Tokyo Chemical Industry Co., Ltd.) was weighed and dissolved in 1 mL of 100 mM 2-morpholinoetanene sulfonic acid (MES) buffer (pH 6.0).
  • MES 2-morpholinoetanene sulfonic acid
  • the reaction solution was subjected to gel filtration chromatography using a PD-10 column (GE Healthcare) using PBS as an elution buffer.
  • the solution after gel filtration was subjected to ultrafiltration using a centrifugal ultrafiltration filter (Amicon Ultra-4 10k; Merck Millipore).
  • the obtained PAA-DADMA-C5-Ph solution was diluted 20-fold, and the diluted solution was used with a microplate (greiner bio-one UV-STAR MICROPALLETE 96 WELL F-BODEN) and a plate reader (TECAN infinite M200 PRO). The absorbance at 386 nm was measured. Then, the concentration of the PAA-DADMA-C5-Ph solution was adjusted with PBS so that the absorbance at 386 nm was about 0.55 when the PAA-DADMA-C5-Ph solution was diluted 20-fold. As the absorbance, the value obtained by subtracting the measured absorbance of PBS as a blank value was used.
  • Example 8 Production of BSA-C5-Ph
  • Ph-C5-Su manufactured by Tokyo Kasei Kogyo Co., Ltd.
  • HEPES 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid
  • bovine serum albumin (BSA) (Nacalai Tesque product code 01860-65; general grade pH 7.0) was weighed and dissolved in 200 ⁇ L of 100 mM HEPES buffer (pH 7.0). I let you. The above two solutions were mixed and reacted at room temperature for 4 hours with stirring.
  • BSA bovine serum albumin
  • the reaction solution was subjected to gel filtration chromatography using PD MiniTrap G-25 (GE Healthcare) using PBS as an elution buffer.
  • the solution after gel filtration was subjected to ultrafiltration using a centrifugal ultrafiltration filter (Amicon Ultra-4 30k; Merck Millipore).
  • the obtained solution of BSA-C5-Ph is diluted 20 times, and the diluted solution is 386 nm using a microplate (greiner bio-one UV-STAR MICROPALLETE 96 WELL F-BODEN) and a plate reader (TECAN infinite M200 PRO). Dilution was measured. Then, the concentration of the BSA-C5-Ph solution was adjusted with PBS so that the absorbance at 386 nm was about 0.55 when the BSA-C5-Ph solution was diluted 20-fold. As the absorbance, the value obtained by subtracting the measured absorbance of PBS as a blank value was used.
  • the reaction solution was subjected to gel filtration chromatography using PD MiniTrap G-25 (GE Healthcare) using PBS as an elution buffer.
  • the solution after gel filtration was subjected to ultrafiltration using a centrifugal ultrafiltration filter (Amicon Ultra-4 30k; Merck Millipore).
  • the obtained GDH-C5-Ph solution is diluted 20-fold, and the diluted solution is 386 nm using a microplate (greiner bio-one UV-STAR MICROPALLETE 96 WELL F-BODEN) and a plate reader (TECAN infinite M200 PRO). Dilution was measured. Then, the concentration of the GDH-C5-Ph solution was adjusted with PBS so that the absorbance at 386 nm was about 0.55 when the GDH-C5-Ph solution was diluted 20-fold. As the absorbance, the value obtained by subtracting the measured absorbance of PBS as a blank value was used. As the absorbance, the value obtained by subtracting the measured absorbance of PBS as a blank value was used.
  • Example 10 Production of GDH-C22-Ph
  • a phenazine derivative 5- ⁇ 11-[11-(2,5-dioxopyrrolidine-1-yloxy) -11-oxoundecylamino] -11-oxoundesyl ⁇ -1-methoxyphenazine-5-ium nitrate (Ph) -C22-Su) (manufactured by Tokyo Chemical Industry Co., Ltd.) was weighed 2.78 mg and dissolved in 500 ⁇ L of ethanol.
  • the reaction solution was subjected to gel filtration chromatography using PD-10 (GE Healthcare) using PBS as an elution buffer.
  • the solution after gel filtration was subjected to ultrafiltration using a centrifugal ultrafiltration filter (Amicon Ultra-4 30k; Merck Millipore).
  • the obtained GDH-C22-Ph solution is diluted 20-fold, and the diluted solution is 386 nm using a microplate (greiner bio-one UV-STAR MICROPALLETE 96 WELL F-BODEN) and a plate reader (TECAN infinite M200 PRO). Dilution was measured. Then, the concentration of the GDH-C22-Ph solution was adjusted with PBS so that the absorbance at 386 nm was about 0.55 when the GDH-C22-Ph solution was diluted 20-fold. As the absorbance, the value obtained by subtracting the measured absorbance of PBS as a blank value was used.
  • the reaction solution was subjected to gel filtration chromatography using a PD-10 column (GE Healthcare) using PBS as an elution buffer.
  • the solution after gel filtration was subjected to ultrafiltration using a centrifugal ultrafiltration filter (Amicon Ultra-4 30k; Merck Millipore).
  • the obtained solution of PLL-C5-Ph_2 was measured with a microplate (greiner bio-one UV-STAR MICROPALLETE 96 WELL F-BODEN) and a plate reader (TECAN infinite M200 PRO), and the absorbance at 386 nm was 0.52 with PBS. Adjusted to the range of ⁇ 0.57. As the absorbance, the value obtained by subtracting the measured absorbance of PBS as a blank value was used.
  • the reaction solution was subjected to gel filtration chromatography using a PD-10 column (GE Healthcare) using PBS as an elution buffer.
  • the solution after gel filtration was subjected to ultrafiltration using a centrifugal ultrafiltration filter (Amicon Ultra-4 30k; Merck Millipore).
  • the obtained PLL-PEG5-Ph solution was measured with a microplate (greiner bio-one UV-STAR MICROPALLETE 96 WELL F-BODEN) and a plate reader (TECAN infinite M200 PRO), and the absorbance at 386 nm was 0.52 with PBS. Adjusted to the range of ⁇ 0.57. As the absorbance, the value obtained by subtracting the measured absorbance of PBS as a blank value was used.
  • Example 13 Production of PLL-PEG13-Ph] 2 mg of Ph-C6-NH2 (manufactured by Tokyo Chemical Industry Co., Ltd.) was weighed and dissolved in 300 ⁇ L of 100 mM MES buffer (pH 6.0). Separately, 3.26 mg of Acid-PEG13-NHS ester (BROADPHARM) was weighed and dissolved in 300 ⁇ L of 100 mM MES buffer (pH 6.0). The above two solutions were mixed and reacted at room temperature for about 20 hours with stirring to obtain a solution B containing a PEG chain-bonded phenazinium nitrate represented by the general formula (10 g) as a phenazine derivative.
  • a PEG chain-bonded phenazinium nitrate represented by the general formula (10 g) as a phenazine derivative.
  • the reaction solution was subjected to gel filtration chromatography using a PD-10 column (GE Healthcare) using PBS as an elution buffer.
  • the solution after gel filtration was subjected to ultrafiltration using a centrifugal ultrafiltration filter (Amicon Ultra-4 30k; Merck Millipore).
  • the obtained PLL-PEG13-Ph solution was measured with a microplate (greiner bio-one UV-STAR MICROPALLETE 96 WELL F-BODEN) and a plate reader (TECAN infinite M200 PRO), and the absorbance at 386 nm was 0.52 with PBS. Adjusted to the range of ⁇ 0.57. As the absorbance, the value obtained by subtracting the measured absorbance of PBS as a blank value was used.
  • Example 14 Production of PLL-PEG25-Ph] 2 mg of Ph-C6-NH2 (manufactured by Tokyo Chemical Industry Co., Ltd.) was weighed and dissolved in 300 ⁇ L of 100 mM MES buffer (pH 6.0). Separately, 5.44 mg of Acid-PEG25-NHS ester (BROADPHARM) was weighed and dissolved in 300 ⁇ L of 100 mM MES buffer (pH 6.0). The above two solutions were mixed and reacted at room temperature for about 20 hours with stirring to obtain a solution C containing a PEG chain-bonded phenazinium nitrate represented by the general formula (10h) as a phenazine derivative.
  • a PEG chain-bonded phenazinium nitrate represented by the general formula (10h) as a phenazine derivative.
  • the reaction solution was subjected to gel filtration chromatography using a PD-10 column (GE Healthcare) using PBS as an elution buffer.
  • the solution after gel filtration was subjected to ultrafiltration using a centrifugal ultrafiltration filter (Amicon Ultra-4 30k; Merck Millipore).
  • the obtained PLL-PEG25-Ph solution was measured with a microplate (greiner bio-one UV-STAR MICROPALLETE 96 WELL F-BODEN) and a plate reader (TECAN infinite M200 PRO), and the absorbance at 386 nm was measured with PBS. Adjusted to the range of 0.52 to 0.57. As the absorbance, the value obtained by subtracting the measured absorbance of PBS as a blank value was used.
  • the obtained Ph-C6-NH2 solution is diluted 20-fold, and the diluted solution is 386 nm using a microplate (greiner bio-one UV-STAR MICROPALLETE 96 WELL F-BODEN) and a plate reader (TECAN infinite M200 PRO). Dilution was measured. Then, the concentration of the Ph-C6-NH2 solution was adjusted with PBS so that the absorbance at 386 nm was about 0.55 when the Ph-C6-NH2 solution was diluted 20-fold. As the absorbance, the value obtained by subtracting the measured absorbance of PBS as a blank value was used.
  • the reagent layer on the working electrode was formed by any of the following (i) to (iii).
  • (I) 10 ⁇ L of each of the solutions of various phenazine derivative-bound high molecular weight redox polymers to which the synthetic high molecular weight polymers obtained in Examples 1 to 7 were bonded was applied onto the working electrode and dried.
  • (Ii) 0.6 ⁇ L of Ketjenblack suspension was applied on the working electrode and dried for about 10 minutes. Then, 0.6 ⁇ L of a solution of various phenazine derivative-binding proteins to which the proteins obtained in Examples 8 to 10 were bound was applied, and the mixture was dried for about 1 hour.
  • (Iii) 10 ⁇ L of the Ph-C6-NH2 solution obtained in Comparative Example 1 was applied onto the working electrode and dried.
  • ⁇ Sensor manufacturing> 0.5 ⁇ L of Ketjenblack suspension was applied onto the gold working electrode and dried for about 5 minutes. Further, coating and drying were repeated twice, and the Ketjen black suspension was applied three times in total. After the reaction for 2 hours, 0.5 ⁇ L of the enzyme / mediator solution was applied and dried for about 30 minutes. Further, the sensor was prepared by immersing it in a P4VP ethanol solution, drying it for 10 minutes, then immersing it again, and drying it for 30 minutes or more to form a protective film.
  • the above sensor is used as the working electrode, the gold electrode is used as the counter electrode, and the potentiostat (BAS Co., Ltd.) is used as the reference electrode with a three-electrode type of Ag / AgCl (saturated potassium chloride) (BAS Co., Ltd.). Then, it was immersed in the prepared sensor PBS and measured using an amperometric it curve. Glucose was added to 50, 150, 300, and 500 mg / dL every 500 seconds from 1000 seconds after the start of measurement, and the current response value was continuously measured. After the measurement, the sensor was stored in PBS at 37 ° C., and the same measurement was performed after 1 day storage and 3 days storage.
  • each measurement data shown in FIG. 13 and Table 3 shows in FIG. 12, 10 seconds before, 20 seconds before, 30 seconds before, 40 seconds before, and 50 seconds before the next glucose addition after the glucose to be measured is added. It is the average value of the measured values of 5 points two seconds ago.
  • the redox mediator phenazine derivative
  • the outflow of the redox mediator to the outside of the protective film can be further prevented or suppressed. it can.
  • storage stability durability

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Abstract

La présente invention concerne un moyen d'inhibition ou de suppression de l'écoulement sortant d'un médiateur redox constituant une couche de réactif dans une sonde d'un biocapteur implantable, en particulier un moyen permettant d'améliorer la stabilité au stockage (durabilité) tout en maintenant la sensibilité de mesure du glucose. Selon la présente invention, le polymère redox de poids moléculaire élevé est représenté par la formule générale (A) [dans la formule, X- représente une espèce anionique, L représente un lieur, poly représente un polymère de poids moléculaire élevé, et R1-R8 représentent chacun indépendamment un atome d'hydrogène ou un substituant.]. Selon la présente invention, ce biocapteur comprend une électrode de travail, une contre-électrode, une couche de réactif disposée sur l'électrode de travail, ainsi qu'un film protecteur recouvrant au moins la couche de réactif, et la couche de réactif comprend une oxydoréductase qui oxyde ou déshydrogène l'analyte et au moins un polymère redox de poids moléculaire élevé représenté par la formule générale (A).
PCT/JP2019/038464 2019-09-30 2019-09-30 Polymère redox de poids moléculaire élevé et biocapteur l'utilisant WO2021064774A1 (fr)

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JP2021550738A JP7285332B2 (ja) 2019-09-30 2019-09-30 高分子量レドックスポリマー及びそれを用いたバイオセンサ
PCT/JP2019/038464 WO2021064774A1 (fr) 2019-09-30 2019-09-30 Polymère redox de poids moléculaire élevé et biocapteur l'utilisant
US17/764,842 US20220322978A1 (en) 2019-09-30 2019-09-30 High molecular weight redox polymer and biosensor using same
CN201980100881.5A CN114450313A (zh) 2019-09-30 2019-09-30 高分子量氧化还原聚合物和使用其的生物传感器

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WO2004058958A1 (fr) 2002-12-24 2004-07-15 Ikeda Food Research Co., Ltd. Glucose dehydrogenase de liaison coenzymatique
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JPH10291368A (ja) 1997-04-17 1998-11-04 Ricoh Co Ltd 光記録媒体
JP2003514924A (ja) 1999-11-15 2003-04-22 セラセンス インコーポレーテッド ポリマー遷移金属錯体及びその用途
WO2004058958A1 (fr) 2002-12-24 2004-07-15 Ikeda Food Research Co., Ltd. Glucose dehydrogenase de liaison coenzymatique
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JP2008228740A (ja) 2006-03-31 2008-10-02 Toyobo Co Ltd アスペルギルス・オリゼ由来グルコースデヒドロゲナーゼ
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WO2010140431A1 (fr) 2009-06-04 2010-12-09 キッコーマン株式会社 Glucose déshydrogénase liée à une flavine
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JP2017517480A (ja) * 2014-04-14 2017-06-29 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ フェナジニウムメディエーター
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